Title: Hox function in Skeletal Stem Cells is critical for propagation of signaling required during fracture repair
Abstract: Hox genes are spatially restricted transcription factors that are critical for patterning the skeleton along the axial-appendicular and proximal-distal skeletal axes. In mammals, Hox genes perform many of their functions redundantly among related paralogous genes; the Hox11 paralogous group genes (HoxPG11) being responsible for patterning the zeugopod (radius/ulna; tibia/fibula) of the limbs and sacrum (Wellik and Capecchi, 2003). Prior studies have revealed that Hoxa11 expression uniquely marks skeletal stem cells (SSCs) and that these genes function in the bone during development, postnatal growth and adult homeostasis (Rux et al., 2016, Pineault et al., 2019, Song et al., 2020). Given these roles, we hypothesize that HoxPG11 genes also function during adult fracture repair. To assess this, we developed an ulnar fracture model, which leads to the simultaneous differentiation of SSCs into the osteogenic and chondrogenic lineages as part of the repair process. HoxPG11 mutants are unable to properly remodel the skeleton in response to injury. Differentiation into the osteoblast lineage is initiated in both control and mutant calluses, but mutant osteoblasts fail to mature. To assess for transcriptional changes between control and HoxPG11 mutant SSCs as they differentiate into osteoblasts and chondrocytes, we performed scRNA-seq on Hoxa11 lineage-labeled cells from control (ROSALSL-td-tomato/+;Hoxa11CreERT2/eGFP;Hoxd11+/+) and mutant (ROSALSL-td-tomato/+;Hoxa11CreERT2/eGFP;Hoxd11loxP/loxP) calluses at 10 days post-fracture (DPF). At 10 DPF, SSCs are rapidly expanding and differentiating into both osteoblasts and chondrocytes through a newly reported injury-induced fibrogenic cell state, characterized by high expression of matrix and signaling activity (Perrin et al., 2024). Control and Hox mutant SSCs are present in similar numbers and cluster together in UMAP visualizations. Likewise, control and Hox mutant lineage cells enter the fibrogenic state, evidenced by the co-expression of lineage cells and identifying matrix markers in the fibrogenic region of the callus. However, Hox mutant fibrogenic cells are signaling deficient compared to their control counterparts, disrupting the amplification of important repair signaling pathways. Loss of this signaling halts the progression of osteoblastogenesis and fracture repair. Thus, our data show that Hox11 function is necessary to coordinate signaling during the early differentiation stages of fracture repair and that disruption of Hox11 function impairs osteoblastogenesis as a result of altered cell-cell communication.
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